Microbiological production of riboflavin



mean ng. 2,1 49 2,477,312

UNITED STATES PATENT OFFICE.

Abraham Levlton, Washington, D. 0., assignor to United States of America as represented by the Secretary of Agriculture t No Drawing. Application September 13, 1946, Serial No. 696,959

3 Claims. (Cl. 195-42) 7 (Granted under the act of March 3, 1883, as amended April 30, 1928; 370 0. G. 757) This application is made under the actof March Naisioi (a reducing agent for H202) potassium 3, 1883, as amended by the act of April 30, 1928, iodide, KI, and yeast dialysate (which may be preand the invention herein described, if patented, pared from bakers yeast by autolysis with ethyl may be manufactured and used by or for the acetate, dialysis of the resulting suspension, and Government of the United States of America for concentration of the resulting dialysate) are governmentalpurposes without the payment to among the chemicals which may be used for the me of any royalty th r on, purpose, the yeast dialysate being preferably em- This invention relates to the microbiological ployed in combination with any of the others. Inproduction of riboflavin by fermentation ofcarstead of yeast dialysate, the equivalent amount of bohydrates with the organism C'lostridium aceto- 10 yeast ,(1 cc. of dialysate equivalent to about .4 gm. butylicum and related strains, andhas among its yeast) may be used. The concentration of yeast objects a process which increases the yield of the dialysate should, in event of use by itself or in riboflavin, combination with the others, be a minimum of In the fermentation process, a trace of ferrous not less than about 1% of the medium, higher iron is usually present Th ir may b d rived ll! concentrations being permissible without deleterifrom the use of ironequipment. It is known that ous efiects. In event of the use of yeast dialysate theiron in certain amounts is desirable or necesalone, iron in concentrations over 0.01 and as high sary for proper utilization of carbohydrates in as about 0.03 mg. atoms per i. of medium may be the process, but that the presence of the iron present. In event yeast be used, the minimum inhibits the production of the riboflavin, whence concentration should be not less than about .4% attempts have been made to regulate the amount of the medium. In event of the use. of yeast diof iron t a concentration t give good utilization ,alysate in combination with the other chemicals,

of carbohydrates without, however; unduly concentrations of the catalase and KI may vary hibiting the production of. the riboflavin, thus to respectively 81001115 from 2.8 10- t0 X obtain the most economical yield of the product. (Preferred, although e upper limit is not criti- According to the prior art processes, the amount cal) and about from 0.03 to 0.60 millimol per 1. 0f of i permissible t permit, synthesis of a gig. medium, with iron in concentration over 0.01 and nificant quantity of riboflavin is limited, in the p o about 0.03 mg. atoms per 1., and concentrarange near 0.01 mg. atoms per 1. of nutritive metlon 0f the Na2s2o4 t y about rom 0.03 to dium as the upper limit. With higher concentra- 015 I11111111101 P L h ron n concentration over tion of iron, more complete utilization of the carand p to about oms per 1. bohydrates may be obtained, but the quantity of To the invention in a detall, a riboflavin produced is not significant. basal medlum was Prepared contammg 0.5 gm.

According to the present invention, it has been MgSo4-7H2O, 03 discovered that the addition of certain chemicals C cmzmo to the nutritive medium containing carbohydrates 21) gm (Nmhso, 29 gm asparagme 30.0 gm.

makes it possible to utilize a higher concentration starch (on dry basis 1X 10-6 gm biotin 50X 10-6 of iron in the medium without inhibiting the propara-aminobenzoic acid, 10 1 yeast dialyduction of the riboflavin or destroying it after its t traces of Zn and and 25 1 of p o or to efiect a muchvhigher i l f 40 either M/5 sodium pyruvate or sodium butyrate agiven concentration of the iron, thus to increase :buifer (pH 6.0) made up to 1 liter with water.

e yield based on the amount o carbohydrates 12.5 ml. portions of this basal medium were disemployed. v persed in anaerobic culture tubes, to which the Crystalline catalase (a specific enzyme for the chemicals under investigation and sufiicient water decomposition of H203), sodium hydrosulflte, to bring the volume in each tube to 15 ml. were the action of the iron is destruction of the riboflavin rather than inhibition of its formation.

In the followng Table II, the tests being run to show the effects of the chemicals indicated, the medium used in each instance contained iron in concentration of .038 mg. atoms per 1., and 13.3 micromoles of riboflavin per 1. was added. The first item of the table compares to the last item of Table I.

tagoni'zed by an increase in iron concentration, and consequently by the use ofthe yeast dialysate with higher concentration of iron, the functions of other added chemicals may be studied, and at the same time normal rates of fermentation achieved.

Each sterilized and cooled tubedescribed above was inoculated with the organism Clostridium acetobutylicum (Weizmann strain No. 4259) a 2% inoculum of cells in 5% corn mash being employed. The inoculum was derived by a series of transfers from a spore culture in corn mash, activated by a heat treatment at 100 C. for one minute. Transfers to the-tubes were made after approximately by weight of available gas had been evolved from the corn mash.

In most tests conducted, riboflavin was added to the medium for the purpose of determining whether the action of the iron was an inhibitory eiifect in the production of the riboflavin or a destructive eifect on the riboflavin after its formation.

Experiments were run to determine the effect of varying amounts of iron in the absence of the chemicals other than the yeast dialysate, the re- Table I shows that using yeast dialysate alone in the medium, iron in concentration of 0.038 mg. atoms per 1. medium is about the upper limit allowable for the production of any riboflavin. At this concentration, substantially no riboflavin is produced and recovered. Also, substantially all added riboflavin is destroyed, thus indicating that Table II chemmlm'bd Total ribo- Added Produced z riboflavin Y; Catalase Nmao. KI recovered I millimole's millimole's millim'oles i per cent per 1. per l. per 1.

none none none 1. 0 0. 8 0. 0

4X10 none none 9. 6 06. 0 0. 0

none 0. 1 none 10. 0 09. 0 0. 0

none none 0. 1 l0. 0 60. 0 0. 0

Table 11 indicates that, under the concentrations of iron and riboflavin present and amounts of chemicals under test, substantially no produced riboflavin is recovered, but that the presence of the chemicals materially decreases the percentage of riboflavin destroyed. Also, the table indicates that the eflects of the three chemicals under test are quite similar.

Table III shows more complete results with catalase and varying concentrations of iron, other factors being similar to those used in the experiments of Tables I and II, using sodium pyruvate buffer (pH 6.0) in the medium.

Table III Produced Fe, mg. Catalase riboflavin t f f f 'f atoms milllmoles recovered, g g per l. per]. miclrgrinoles W evolved 0218 none 18. 8 l0 0254 none 11. 7 30 0290 none 6. l 60 0344 none 1. 6 70 0Z1) 2.8X10' 40. 0 5O 0290 2.8Xl0" 27. 0 l. 00

.0344 2.8)(10 1.3 LIX) Table III exhibits that in concentrations of iron ranging up to about 0.0300 mg. atoms per 1. medium, the presence of catalase in the concentration indicated increases the yield of riboflavin about 250%. More catalase may be used than that indicated, but this will not substantially increase the riboflavin production.

Table IV shows more complete results with varying amounts of N32S2O4 and K1 at 0.033 mg.

atoms of iron per 1. medium (near the critical concentration of the iron), the medium being similar to that used in the experiments of Table at least with the hisher concentrations of iron, 111, but using sodium butyrate butler.

Table IV indicates that at concentrations of iron at or near the critical point, high concentrations of NazSzOr prevent the production and recovery of any riboflavin, but when used in lower concentrations, the yield of riboflavin is increased in an amount comparable to the 250% increase found with the use of catalase. Also, at these high concentrations of iron, KI actually contributes to the destructive action of the iron. In general, therefore, NaaSaOa in concentrations less than approximately 0.5 millimole per liter. extendsthe range of iron concentration beyond 0.033 mg. atoms per liter. KI does not, however, share this efiect with NaaS2O4.

In addition to the chemicals above considered, it has been found that yeast dialysate also counteracts the destructive action of iron in the fermentation production of riboflavin. To exhibit this, a basal medium was prepared containing 0.5 gm. KH2P04, 0.5 gm. MgS04.7H20, 0.3 gm. CaCla2H2O, 2.0 gm. (NH4)2S04, 2.0 gm. asparagine, 30.0 gm. lactose, 1.6X10- gm. potassium iodide, 2.75 gm. sodium lactate, 1x10 gm. biotin and 50 10'- gm. para-aminobenzoic acid made up to one liter with water.

Iron was added to the medium to give a total I of 0.011 mg. atoms of iron per 1., and inoculation was carried out. in a manner similar to that before indicated. Without the addition of any yeast dialysate, no significant quantity of ribo- 6' the various chemicals before mentioned comparable to the conditions in the fermentation process) parallels very closely the results obtained in the fermentation process. 1

With this understanding, it will be noted that catalase and NazS204 are quite similar in that they both decompose H202 to water and thus probably act to remove the H202 before it has opportunity together with the iron to destroy the riboflavin.

Spectrophotometric studies indicate that the action of H20 and Fe takes place at position 3 in the structural formula for riboflavin given flavin was produced, but with the addition of 1% yeast dialysate (about .4% yeast), a yield of 40 micromoles of riboflavin per 1. of medium was obtained. Even with much higher concentrations of iron (up to 0.03 mg. atoms per 1.), similar its'existence in the transitory state only. Riboflavin itself is quite stable in the-presence either of iron solutions or of H202, but tests show that ferrous iron and HzOatogether are destructive of riboflavin. Furthermore, the tests show that the destructive action of riboflavin with H20: present (using riboflavin in aqueous solution with conditions of iron concentration and tion of what is flavin by fermentation with Clostridium acetobutylicum of a nutritive mediumcontaining carbohydrates and iron, increasing the yield-of riboflavin based on the amount of carbohydrates employed by adding to the nutritive, medium yeast and catalase.

2. In the microbiological production of riboflavin by fermentation with Clostridium aceto butylicum of a nutritive medium containing carbohydrates and iron, increasing the yield of riboflavin based on the amount of carbohydrates employed by adding to the nutritive medium yeast.

3. In the microbiological production of riboflavin by fermentation with Clostridium acetobutylicum of a nutritive medium containing carbohydrates and iron, increasing the yield of riboflavin based on the amount of carbohydrates employed by adding to the nutritive medium catalase.

ABRAHAM LEVITON.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PA'I'ENTS Sumner et al., Enzymes, Academic Press (1943) pages 176 to 1'18, Book 513.5.

Leviton, J. A. C. S. 68, 5, May 1946, pp. 835 to 840. 

